Members of the ligand-induced nuclear receptor superfamily are medically important regulators of cellular activity. Hormones initiate a receptor activation process leading to receptor binding to specific DNA recognition sites in the promoter regions of their target genes. Using microscopy and fluorescent protein chimeras of nuclear receptors, we pioneered studies establishing that ligand binding regulates the subcellular targeting of glucocorticoid (GR), vitamin D (VDR), and retinoid X receptors (RXR). We have continued to use advanced microscopy techniques to address the roles of receptor trafficking in hormone actions.We demonstrated that heterodimerization with RXR has a profound effect on VDR subcellular distribution and that this effect is physiologically important. When expressed separately, the steady-state distribution of the YFP-RXR was more nuclear than the GFP-VDR. Experiments measuring fluorescence recovery after photobleaching (FRAP) and fluorescence loss in photobleaching (FLIP) demonstrated that both VDR and RXR constantly shuttle between the cytoplasm and the nucleus and that RXR shuttles at a faster rate than VDR. Coexpression of RXR-BFP with GFP-VDR promoted nuclear accumulation of the later by influencing both nuclear import and retention. RXR-BFP also promoted hormone-dependent nuclear accumulation of a nuclear localization signal mutant receptor (nlsGFP-VDR), and rescued its transcriptional activity. Heterodimerization mutant RXR failed to alter GFP-VDR and nlsGFP-VDR distribution or activity. This finding on the ability of RXR to facilitate the nuclear import of VDR is most likely applicable for other heterodimerization partners of RXR, such as the thyroid hormone receptor and the retinoic acid receptor. We and others have observed that ligand binding induces formation of multiple nuclear foci of GFP-GR, GFP-VDR, YFP-RXR, GFP-ER but the physiological importance of these foci remained to be elucidated. Studies on a cell line harboring a large array of MMTV-LTR allowed direct observation of human GR binding to this array. Mutational analysis demonstrated a correlation between hormone-dependent nuclear foci formation and DNA binding for both the VDR and the GR. In addition, fluorescence energy transfer experiments (FRET) revealed that calcitriol induces intranuclear foci formation of VDR/RXR heterodimers. Because VDR and RXR bind to DNA as heterodimers, this finding also suggest a correlation between focal receptor accumulation and DNA-binding. Current efforts are directed towards characterizing this and other subnuclear domains involved in nuclear receptor targeting.